Genomic DNA molecules contain methylated bases in vivo. Such methylation affects a number of physiological functions, such as gene expression. DNA Methylation also carries important pathological consequences, which explains why it became an important research topic in recent years. For example, studies have found that DNA methylation levels differ between normal tissue and cancer tissue. In addition to its implication in cancer, DNA methylation also plays important roles in epigenetic inheritance, embryonic development, postnatal development, as well as bacteria host defense.
Given the importance of DNA methylation, it is crucial to accurately measure the methylation level of a given genomic DNA, or compare the methylation levels between genomic DNA samples. The large size of genomic DNA is difficult to manipulate and study as a whole. The digestion of genomic DNA to smaller pieces by restriction enzymes is one of the commonly used methods. The DNA fragment mixture after restriction enzyme digestion is defined as restriction library. Accurate measurement of genomic DNA methylation level can be reached through accurately tagging the restriction library and subsequently amplifying them in a faithful fashion. Methylation Sensitive Restriction Enzyme (MSRE) play an important role in the analysis of DNA methylation, but traditional methods of tagging the restriction library suffer because of the inaccuracies in ligation process, which is mainly caused by the self-ligation of the restriction fragments. There exists an urgent need for a better and more accurate method of breaking down and amplifying the genomic DNA, as well as accurately measuring the methylation level of one genomic DNA, or comparing the methylation levels between two genomic DNA samples.